Epitopes in viral envelope proteins and specific antibodies directed against these epitopes: use for detection of HCV viral antigen in host tissue

ABSTRACT

Antibodies to two new epitopes on the HCV envelope proteins were identified which allow routine detection of native HCV envelope antigens, in tissue or cells derived from the host. The new epitopes are: the E1 region aa 307-326 and the N-terminal hyper variable region of E2 aa 395-415. Surprisingly, we characterised an antibody that reacts with various sequences of the hypervariable domain of E2. Specific monoclonal antibodies directed against these epitopes and allowing routine detection of viral antigen are described.

FIELD OF THE INVENTION

[0001] The present invention is based on the finding that antibodiesdirected against specific epitopes of the E1 and E2 protein of HCV canbe used to detect viral antigens in host tissues.

BACKGROUND OF THE INVENTION

[0002] Hepatitis C virus (HCV) infection is a major health problem inboth developed and developing countries. It is estimated that about 1 to5% of the world population is affected by the virus. HCV infectionappears to be the most important cause of transfusion-associatedhepatitis and frequently progresses to chronic liver damage. Moreover,there is evidence implicating HCV in induction of hepatocellularcarcinoma. Consequently, the demand for reliable diagnostic methods andeffective therapeutic agents is high. Also sensitive and specificscreening methods of HCV-contaminated blood-products and improvedmethods to culture HCV are needed.

[0003] HCV is a positive stranded RNA virus of approximately 9,400 baseswhich encode at least three structural and six non-structural proteins.Based on sequence homology, the structural proteins have beenfunctionally assigned as one single core protein and two envelopeproteins: E1 and E2. The E1 protein consists of 192 amino acids andcontains 5 to 6 N-glycosylation sites, depending on the HCV genotype.The E2 protein consists of 363 to 370 amino acids containing 9 to 11N-glycosylation sites, depending on the HCV genotype (for review seeMajor and Feinstone, 1997; Maertens and Stuyver, 1997). The E1 proteincontains various variable domains, while the E2 protein contains twohypervariable domains, of which the major domain is located at theN-terminus of the protein (Maertens and Stuyver, 1997). The envelopeproteins have been produced by recombinant techniques in Escherichiacoli, insect cells, yeast cells and mammalian cells. The usage of anexpression system in higher eukaryotes and especially in mammalian cellculture leads to envelope proteins of superior quality, i.e. they areeffectively recognised by antibodies in patient samples as described inPCT/EP 95/03031 to Maertens et al.

[0004] Currently, the detection of HCV in cells or tissues relies mainlyon the demonstration of viral RNA. However, RNA detection in cells ortissues is a cumbersome technique which either involves the extractionof RNA followed by reverse transcription and nested PCR or includes insitu RT-PCR and hybridisation. Since these techniques are also prone tofalse positive reactivity, viral RNA detection is exclusively performedon serum samples. Reliable methods for the detection of viral proteinantigens in serum and tissue samples are still lacking.

[0005] The replication sites of HCV have not yet been fully elucidated.It is generally accepted that the virus replicates in hepatocytes, butreplication in other tissues, such as lymphoid tissues, is still highlydebated. Hence, a reliable method for the detection of viral proteins orthe virus itself in cells may solve this issue.

[0006] The detection of viral proteins in cells has been hampered by thelack of antibodies which specifically bind to viral proteins and whichare able to specifically recognise the natural HCV protein antigens(i.e. antigens as expressed by the host following infection with HCV).As a consequence, to date only few studies relate to demonstrating thepresence of viral HCV proteins in host cells (for review see Guido andThung, 1996). Moreover, host-derived antibodies have been used in manyof these studies. Preparations containing host-derived antibodies,however, cannot be reproduced easily and these preparations may becontaminated by autoimmune antibodies as well as by antibodies againstother known or even unknown agents. In contrast, it is known thatantibodies produced in animals upon immunisation with recombinantantigens will yield antibodies with the desired specificity. In order tohave reproducible quality, monoclonal antibodies are preferred as well.In addition, the envelope proteins of HCV need to be produced by amammalian expression system to yield good quality antigens. Currently,this expression condition goes together with incomprehensible problems.Therefore, only few monoclonal antibodies have been described whichcould be used to detect HCV envelope antigens in tissue specimens ofpatients. These antibodies were directed against the N-terminal regionof E1, amino acids (aa) 192-226, (Hiramatsu et al., 1992, Kaito et al.,1994) or the C-terminal domain of E2, aa 451-715 (Sansonno et al.,1997a, 1997b). However, from these publications and from the reviews byGuido and Thung (1996), and Liang (1996) it is evident that there isstill an existing need for well-characterised antibodies allowingefficient and routine detection of natural HCV protein antigens in serumand tissue samples. This need was recently confirmed by a study by Driesand co-workers (1999). Dries' group proved that 61% of HCV antibodypositive and serum RNA negative individuals were carriers of HCV as HCVRNA could be detected in liver biopsy samples but only one third ofthese cases could be detected by immunohistochemistry using a panel ofantibodies. Thus, routine detection of HCV antigen in liver biopsiesstill lacks sensitivity. Also the detection of viral antigens in bodyfluids such as serum or plasma is hampered by the same problem. Up todate only one single technique has been described allowing detection ofcore antigen in these fluids. However, in order to detect the coreprotein, the technique requires a complete denaturation of the coreprotein present in the sample using sodium hydroxide (Kashiwakuma etal., 1996).

[0007] Taken together, the identification of new specific HCV epitopeswhich are accessible for antibodies and which allow antigen detection intissue or body fluid samples is therefore urgently needed. The HCVenvelope proteins are putative candidate targets to find such epitopes,since these proteins should be present in all biological samples: influids, on the membrane of the virus, and in cells from the earliestevent of infection (i.e. viral entry) throughout the completereplication cycle. However, these envelope proteins are highly variableso antibodies with a high cross-reactivity towards the differentgenotypes of HCV are needed. Thus, identification of such epitopes andthe search for antibodies with high cross-reactivity towards thesequence variation of HCV is a challenging undertaking.

[0008] The present application relates to specific monoclonalantibodies, directed against particular epitopes in the envelopeproteins of HCV, which are able to detect HCV antigen in tissuespecimens of patients. In total two such epitopes, and correspondingantibodies, were found: one in the C-terminal region (aa 227-383) of theenvelope protein E1 and one in the N-terminal hypervariable region (HVR)of E2 (aa 384-450). Although the latter region, and more specificallythe region 395-415, is considered to be hypervariable, we characterised,to our surprise, an antibody which reacts with various known sequencesof the HVR of E2.

AIMS OF THE INVENTION

[0009] It is clear from the literature that there is an urgent need todevelop reliable diagnostic methods, reliable vaccines and effectivetherapeutic agents for HCV. Also sensitive and specific screeningmethods of HCV-contaminated blood-products and improved methods toculture HCV are needed. New antibodies able to detect the virus inanimal- or in vitro models, or in its natural host, may help indesigning efficient diagnostic tools and therapeutic agents. In thisregard, the present invention is based on the surprising finding ofmonoclonal antibodies directed against either E1 or E2-HVR which can beused for the detection of HCV antigens in various tissues or cells.These tissues include the liver but also cells derived from bloodsamples. Therefore, the present invention aims at providing and using anantibody specifically binding to HCV envelope protein region aa 227450,which covers the main part (C-terminal) of the E1 protein, and theN-terminal region of the E2 protein. These antibodies allow detection ofnatural HCV protein antigens, and can be used for the preparation of anatural HCV protein antigen detection kit. Notably, the complete E1protein corresponds to aa 192-383, while the complete E2 proteincorresponds to aa 384-747 (see: Major and Feinstone, 1997; Maertens andStuyver, 1997).

[0010] More specifically, the present invention aims at providing andusing an antibody as defined above which specifically binds to at leastone of the following epitopes:

[0011] aa 307-326 of HCV E1 protein (SEQ ID 30)

[0012] aa 395-415 of HCV E2 protein (SEQ ID 31).

[0013] Moreover, the present invention aims at providing and using anantibody as defined above which is a monoclonal antibody. In thisregard, the present invention aims at providing and using a monoclonalantibody secreted by the hybridoma line with ECACC deposit havingaccession number 98031215 or 98031214.

[0014] It should be clear that the present invention also aims atproviding and using any functionally equivalent variant or fragment ofany antibody as defined above, as well as mutant forms thereof, ormolecules exhibiting similar functional binding reactivities with SEQ ID30 and 31, such as sequences obtained from phage- or other libraries.

[0015] In addition, the present invention aims at providing and using ahybridoma cell line secreting a monoclonal antibody which specificallybinds to HCV E1 protein (aa 227-383) or HCV E2 N-terminal hypervariableregion (aa 384450) and which allows the detection of natural HCV proteinantigens, and can be used for the preparation of a natural HCV proteinantigen detection kit. More specifically, the present invention aims atproviding and using the hybridoma cell line corresponding to the ECACCdeposit having accession number 98031215 or 98031214.

[0016] Furthermore, the present invention also aims at providing amethod for the detection of natural HCV protein antigens comprising:

[0017] contacting a test sample which may contain HCV protein antigenswith an antibody as defined above or with a functionally equivalentvariant or fragment of said antibody, to form an antibody-antigencomplex, and

[0018] determining said antigen-antibody complex with an appropriatemarker.

[0019] More specifically, the present invention aims at providing amethod as defined above wherein said test sample comprises human cells,such as peripheral blood cells, or human tissues, such as liver tissue.

[0020] Finally, the present invention aims at providing an assay kit forthe detection of natural HCV protein antigens comprising:

[0021] an antibody as defined above, or, a functionally equivalentvariant or fragment of said antibody, and

[0022] appropriate markers which allow to determine the complexes formedbetween HCV protein antigens in a test sample with said antibody or afunctionally equivalent variant or fragment of said antibody.

[0023] All the aims of the present invention are considered to have beenmet by the embodiments as set out below.

BRIEF DESCRIPTION OF TABLES AND DRAWINGS

[0024] Table 1 provides the peptide sequences of all peptides mentionedin this application.

[0025] Table 2 shows the cross-reactivity of IGH 222 towards varioussequences of the hyper-variable domain in E2. All peptides werebiotinylated, bound to streptavidin-coated microtiterplates and allowedto react with IGH 222.

[0026]FIG. 1 shows staining of E2 antigen, revealed by the monoclonalantibody IGH 222, on a liver biopsy of an HCV patient.Immunohistochemistry was performed on 4 μm thick cryostat sections offresh frozen materials (the liver biopsy was snap-frozen in liquidnitrogen-cooled isopentane and stored at −70° C. until use) using athree step indirect immuno-peroxidase procedure. Sections were incubatedovernight at 4° C. with monoclonal antibody IGH 222 (purified IgG₁: 10ng/μl). The secondary and tertiary antibodies consisted ofperoxidase-conjugated rabbit anti-mouse and peroxidase-conjugated swineanti-rabbit IgG, respectively (both obtained from Dakopatts, Copenhagen,Denmark; working dilution 1/50 and 1/100, respectively). Each incubationwas performed 30 minutes at room temperature and followed by a wash inthree changes of phosphate buffered saline, pH 7.2. The reaction productwas developed by incubation for 15 minutes in 100 mM acetate buffer (pH5.2), containing 0.05% 3-amino-9-ethyl-carbazole and 0.01% H₂O₂,resulting in bright red staining of immuno-reactive sites. The sectionswere counterstained with haemotoxylin. Controls (not shown) consisted ofirrelevant monoclonal antibodies of similar isotype as the primaryantibody, or of chromogen alone: these controls were consistentlynegative. The photograph shows a 25× magnification. The darkest stainingreveals the presence of HCV antigen in hepatocytes only (see arrows).

[0027]FIG. 2A shows staining of E1 antigen, revealed by the monoclonalantibody IGH 207, on a liver biopsy of an HCV patient. The procedurefollowed is identical to the one described in FIG. 1, except for theconcentration of the monoclonal antibody which was 30 ng/μl. Thephotograph shows a 10× magnification on which the staining of the cellsin the lymphocyte infiltrates is dominant. The darkest staining revealsthe presence of HCV antigen in hepatocytes (see arrow) and ininfiltrating lymphocytes (see double arrow).

[0028]FIG. 2B shows staining of E1 antigen, revealed by the monoclonalantibody IGH 210, on a liver biopsy of an HCV patient. The liver biopsywas fixed with formaldehyde and embedded in paraffin. Sections werepretreated by heat (microwave method) and by protease digestion. Theconcentration of the antibody was 6 ng/μl. The photograph shows aclearly stained isolated mononuclear cell (arrow) in a field ofhepatocytes, which do not stain with this monoclonal.

[0029]FIG. 3 shows staining, revealed by the monoclonal antibody IGH207, of intracellular E1 antigen in peripheral blood mononuclear cells.Peripheral white blood cells (0.5×10⁶) were suspended in 200 μl PBS-0.1% saponin, 2 μg of IGH 207 was added and allowed to react for 25minutes at 4° C. The cells are washed three times with 3 ml PBS-0.1%saponin and three times with PBS-0.2% NaN₃. Finally cells areresuspended in 250 μl of PBS-0.2% NaN₃ and analysed by flow cytometry.After gating on the mononuclear cell fraction (right column), thefluorescence was plotted (left column). Samples 1-5 are derived from HCVchronic carriers while sample 6 is derived from a healthy blood carrier.The left column shows two examples of the gating on the mononuclear cellfraction (samples 2 and 6), while the right column shows thefluorescence found in these mononuclear cells. While the control sampleshows no staining at all with this monoclonal antibody, there is amarked positive signal in all HCV patients, except for patient 4 forwhom a weaker signal was obtained.

[0030]FIG. 4 shows staining, revealed by the monoclonal antibody IGH201, of intracellular E1 antigen in peripheral blood mononuclear cells.The technique was similar as described for FIG. 3. Samples 7-11 arederived from HCV chronic carriers while sample 12 is derived from ahealthy blood carrier. The left column shows two examples of the gatingon the mononuclear cell fraction (samples 7 and 12), while the rightcolumn shows the fluorescence found in these mononuclear cells. Althoughthe control sample reveals a higher background staining, the reaction inthe patient samples can be easily discriminated based on the twopopulations which can be detected: a population with a similar stainingas in the control and a second population with high intensity staining,not seen in the control.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The invention described herein draws on previously published workand pending patent applications. By way of example, such work consistsof scientific papers, patents or pending patent applications. All thesepublications and applications, cited previously or below are herebyincorporated by reference.

[0032] It is clear that the detection of viral proteins has beenhampered by the lack of antibodies which specifically bind to viralproteins and which are able to recognise the natural HCV proteinantigens (i.e. protein antigens as expressed by the host followinginfection with HCV). The present invention is based on the finding oftwo new epitopes on the HCV envelope proteins which allow routinedetection of natural HCV protein antigens, by means of antibodiesdirected against these epitopes, in biological samples derived from thehost. Thus, the present invention relates to the use of antibodiesspecifically binding to the C-terminal region of HCV E1 protein (aa227-383) or the N-terminal region of the HCV E2 protein (aa 384-450) forthe preparation of a natural HCV protein antigen detection kit. The term“antibodies specifically binding to the C-terminal region of the HCV E1protein (aa 227-383) or the N-terminal region of the HCV E2 protein (aa384-450)” refers to any polyclonal or monoclonal antibody binding to anhepatitis C viral particle or any molecule derived from said viralparticle, more particularly to the C-terminal region of the E1 proteinand the N-terminal region of the E2 protein. The “envelope region” ofthe HCV viruses, and thus “the C-terminal region of the HCV E1 protein(aa 227-383) or the N-terminal region of the HCV E2 protein (aa 384450)”are well-known regions by a person skilled in the art (Wengler, 1991;Major and Feinstone, 1997; Maertens and Stuyver, 1997).

[0033] The term “bind” indicates that the antibodies of the presentinvention are physically connected to HCV proteins. In particular, thatthe antibodies specifically bind to the HCV envelope proteins, implyingthat there is substantially no cross-reaction with other components ofHCV or other proteins. Binding of the antibody to HCV proteins can bedemonstrated by any method or assay known in the art, such as binding-,ELISA-, and RIA-type of assays or competition assays (e.g. see Currentprotocols in immunology). It should be clear that the region of an HCVenvelope protein which binds to an antibody need not to be composed of acontiguous sequence.

[0034] The term “monoclonal antibody” used herein refers to an antibodycomposition having a homogeneous antibody population. The term is notlimiting regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. In addition,the term “antibody” also refers to humanised antibodies in which atleast a portion of the framework regions of an immunoglobulin arederived from human immunoglobulin sequences and single chain antibodiesas described in U.S. Pat. No. 4,946,778 and to fragments of antibodiessuch as F_(ab), F_(′(ab)2), F_(v), and other fragments which retain theantigen binding function and specificity of the parent antibody. Alsoincluded in the term “antibody” are diabodies, triabodies andtetravalent antibodies, as described in EP application N^(o) 97870092.0to Lorré et al., which retain the antigen binding function andspecificity of the parent antibody. It should be clear that theantibodies as described in the present invention could be used in amethod for the detection of natural HCV protein antigens.

[0035] The term “test sample” refers to any sample obtained from a host,such as serum, plasma, saliva, mucus, spinal cord fluid, or biopsies. Inthis regard, the terms “test sample” and “biological sample” are usedinterchangeable herein. The term “biopsy” particularly refers to asample comprising cells, in particular human cells. Moreover, the latterterm refers also to a sample derived from liquid tissue, such asperipheral blood cells, or from solid tissue, such as liver tissue. Incase antigen detection is performed on a biopsy, the term “in situdetection” is used.

[0036] The term “natural HCV protein antigen detection kit” refers to akit for the detection of any HCV protein antigen, preferentially to thedetection of the C-terminal region of the HCV E1 protein (aa 227-383) orthe N-terminal region of the HCV E2 protein (aa 384-450) in theirnatural or original position, preferentially the antigens as present ina test sample, by any method known to the man skilled in the art. Inother words, the latter term refers to the visualisation of the presenceof the C-terminal region of HCV E1 (aa 227-383) protein or theN-terminal region of the HCV E2 protein (aa 384450) in, or on, theirnatural host cell or tissue by binding of the antibodies of the presentinvention. The detection kit comprises the following components:

[0037] (i) when appropriate a means for isolating a test sample,

[0038] (ii) possibly a solution to permeabilise cells,

[0039] (iii) an antibody as described herein, or a functionallyequivalent variant or fragment thereof,

[0040] (iv) possibly secondary and possibly tertiary antibodies,

[0041] (v) possibly incubation and/or washing buffers,

[0042] (vi) possibly a staining solution.

[0043] Preferentially, said kit is used for the in situ detection of HCVenvelope proteins.

[0044] The natural host cell or tissue can be any host cell or tissuederived from any host species. In particular, the natural host cellrefers to peripheral blood cells and the natural tissue refers to livertissue (see also Examples section of the present application). Thenatural host refers, in particular, to humans but may also refer tonon-human primates or other mammals.

[0045] More specifically, the present invention relates to antibodies,which specifically bind to at least one of the following epitopes: aa307-326 of HCV E1 protein and aa 395-415 of HCV E2 protein. The latterantibodies are secreted by hybridomas deposited at the EuropeanCollection of Cell Cultures (ECACC), Centre for Applied Microbiology &Research, Salisbury, Wiltshire SP4 OJG, U.K. (Tel: +44 1980 612512; fax:+44 1980 611315) on Mar. 13, 1998, which have been assigned thefollowing accession numbers: 98031215 for hybridoma 17H10F4D10 secretingmAb IGH 222, which binds epitope aa 395-415 (SEQ ID 31), and, 98031214for hybridoma 14H11B2 secreting mAb IGH 207, which binds epitope aa307-326 (SEQ ID 30). In this regard, it should be clear that antibodiesthat bind to parts of said epitopes are also part of the presentinvention. For example, antibodies binding to the following epitopes arehereby also included: aa307-311, aa308-312, aa309-313, aa310-314,aa311-315, aa312-316, aa312-317, aa313-318, aa314-319, aa315-320,aa316-321, aa317-322, aa318-323, aa319-324, aa320-325, aa321-326,aa307-312, aa308-313, aa309-314, aa310-315, aa311-316, aa312-317,aa313-318, aa314-319, aa315-320, aa316-321, aa317-322, aa318-323,aa319-324, aa320-325, aa321-326, aa 307-313, aa308-314, aa309-315,aa310-316, aa311-317, aa312-318, aa313-319, aa314-320, aa315-321,aa316-322, aa317-323, aa318-324, aa319-325, aa320-326, aa307-314,aa308-315, aa309-316, aa310-317, aa311-318, aa312-319, aa313-320,aa314-321, aa315-322, aa316-323, aa317-324, aa318-325, aa319-326,aa307-315, aa308-316, aa309-317, aa310-318, aa311-319, aa312-320,aa313-321, aa314-322, aa315-323, aa316-324, aa317-325, aa318-326,aa307-316, aa308-317, aa309-318, aa310-319, aa311-320, aa312-321,aa313-322, aa314-323, aa315-324, aa316-325, aa317-326, aa307-317,aa308-318, aa309-319, aa310-320, aa311-321, aa312-322, aa313-323,aa314-324, aa315-325, aa316-326, aa307-318, aa308-319, aa309-320,aa310-321, aa311-322, aa312-323, aa313-324, aa314-325, aa315-326,aa307-319, aa308-320, aa309-321, aa310-322, aa311-323, aa312-324,aa313-325, aa314-326, aa307-320, aa308-321, aa309-322, aa310-323,aa311-324, aa312-325, aa313-326, aa307-321, aa308-322, aa309-323,aa310-324, aa311-325, aa312-326, aa307-322, aa308-323, aa309-324,aa310-325, aa311-326, aa307-323, aa308-324, aa309-325, aa310-326,aa307-324, aa308-325, aa309-326, aa307-325, aa308-326, and, aa395-399,aa396-400, aa397-401, aa398-402, aa399-403, aa400-404, aa401-405,aa402-406, aa403-407, aa404-408, aa405-409, aa406-410, aa407-411,aa408-412, aa409-413, aa410-414, aa411-415, aa395-400, aa396-401,aa397-402, aa398-403, aa399-404, aa400-405, aa400-406, aa402-407,aa403-408, aa404-409, aa405-410, aa406-411, aa407-412, aa408-413,aa409-414, aa410-415, aa395-401, aa396-402, aa397-403, aa398-404,aa399-405, aa400-406, aa401-407, aa402-408, aa403-409, aa404-410,aa405-411, aa406-412, aa407-413, aa408-414, aa409-415, aa395-402,aa396-403, aa397-404, aa398-405, aa399-406, aa400-407, aa401-408,aa402-409, aa403-410, aa404-411, aa405-412, aa406-413, aa407-414,aa408-415, aa395-403, aa396-404, aa397-405, aa398-406, aa399-407,aa400-408, aa401-409, aa402-410, aa403-411, aa404-412, aa405-413,aa406-414, aa407-415,aa395-403, aa396-404, aa397-405, aa398-406,aa399-407, aa400-408, aa401-409, aa402-410, aa403-411, aa404-412,aa405-413, aa406-414, aa407-415, aa395-404, aa396-405, aa397-406,aa398-407, aa399-408, aa400-409, aa401-410, aa402-411, aa403-412,aa404-413, aa405-414, aa406-415, aa395-405, aa396-406, aa397-407,aa398-408, aa399-409, aa400-410, aa401-411, aa402-412, aa403-413,aa404-414, aa405-415, aa395-406, aa396-407, aa397-408, aa398-409,aa399-410, aa400-411, aa401-412, aa402-413, aa403-414, aa404-415,aa395-407, aa396-408, aa397-409, aa398-410, aa399-411, aa400-412,aa401-413, aa402-414, aa403-415, aa395-408, aa396-409, aa397-410,aa398-411, aa399-412, aa400-413, aa401-414, aa402-415, aa395-409,aa396-410, aa397-411, aa398-412, aa399-413, aa400-414, aa401-415,aa395-410, aa396-411, aa397-412, aa398-413, aa399-414, aa400-415,aa395-411, aa396-412, aa397-413, aa398-414, aa399-415, aa395-412,aa396-413, aa397-414, aa398-415, aa395-413, aa396-414, aa397-415,aa395-414 and 396-415,

[0046] The present invention also relates to functionally equivalentvariants or fragments of the above-indicated antibodies. The term“functionally equivalent variants or fragments” refers to any variant orfragment known in the art of said antibodies which retain the antigenbinding function and specificity of the parent antibody.

[0047] More specifically, the latter term refers to humanised antibodiesand single chain antibodies as defined above and to fragments ofantibodies such as F_(ab), F_(′(ab)2), F_(v), and the like. Alsoincluded are diabodies, triabodies and tetravalent antibodies, asdescribed above, as well as mutant forms thereof or molecules exhibitingsimilar functional binding reactivities with SEQ ID 30 and 31, such assequences obtained from phage- or other libraries as described byLadner, 1995; Maclennan, 1995 and Cannon et al., 1996. Indeed, anypeptide described by these authors, constrained or not, which allows thedetection of natural HCV protein antigens is part of the presentinvention.

[0048] The present invention also relates to hybridoma cell linessecreting antibodies as defined above. In this regard, it should beclear that the hybridoma technology for obtaining mAbs is well known toa person skilled in the art, e.g. essentially according to Kohler andMilstein (1975). It should also be clear that mapping the epitopes towhich the mAbs specifically bind can be performed by any method known inthe art such as the ones described in PCT/EP 97/07268 to Depla et al.

[0049] The present invention also relates to a method for the detectionof natural HCV protein antigens comprising:

[0050] contacting a test sample which may contain HCV protein antigenswith an antibody as defined above or with a functionally equivalentvariant or fragment of said antibody, to form an antibody-antigencomplex, and

[0051] determining said antigen-antibody complex with an appropriatemarker.

[0052] Preferentially, said method is used for the in situ detection ofHCV envelope proteins.

[0053] The term “determining said antigen-antibody complex with anappropriate marker” refers to any method known in the art which detects,or visualises, the above-indicated antigen-antibody complexes, such asfluorescence flow cytometry, binding-, ELISA- and RIA-type assays orcompetition assays (see Examples section, Hertogs et al., 1993, and, WO93/04084 to Mehta et al.). Similarly, the term “appropriate marker”refers to any marker known in the art such as the ones described in WO93/04084 to Mehta et al. and Coligan et al., 1992 which visualises theabove-indicated antigen-antibody complexes.

[0054] In this regard, the present invention also relates to an assaykit for the detection of natural HCV protein antigens comprising: anantibody as defined above, or, a functionally equivalent variant orfragment of said antibody, and appropriate markers which allow todetermine the complexes formed between HCV protein antigens in a testsample with said antibody or a functionally equivalent variant orfragment of said antibody.

[0055] The present invention will now be illustrated by reference to thefollowing examples that set forth particularly advantageous embodiments.However, it should be noted that these embodiments are merelyillustrative and can not be construed as to restrict the invention inany way.

EXAMPLES Example 1 Generation of Monoclonal Antibodies Against E1 and E2

[0056] Mice were immunised with truncated versions of E1 (aa 192-326)and E2 (aa 384-673), expressed by recombinant vaccinia virus, asdescribed in PCT/EP 95/03031 to Maertens et al. After immunisationsplenocytes of the mice were fused with a myeloma cell line. Resultinghybridomas secreting specific antibodies for E1 or E2 were selected bymeans of ELISA.

Example 2 Selection of Monoclonal Antibodies

[0057] A large series (25 in total, of which 14 in detail; the lattercan be obtained from Innogenetics NV, Gent, Belgium) of monoclonalsdirected against E1 or E2 was evaluated for staining of native HCVantigen in liver biopsies of HCV patients and controls (see below).Staining was performed either on cryosections or on formaldehyde fixedbiopsies (for protocols, see figure legends). Only three monoclonalantibodies revealed a clear and specific staining. All other monoclonalantibodies either gave no or very weak staining, or showed non-specificstaining. Remarkably, two different antigen staining patterns werenoticed. Monoclonal IGH 222, directed against E2, clearly stainedhepatocytes (FIG. 1), while IGH 207 and IGH 210, directed against E1,stained lymphocytes infiltrating in the liver (FIGS. 2a and 2b). IGH 207also stained hepatocytes, but to a weaker degree than IGH 222 (compareFIGS. 1 and 2a). This staining pattern was confirmed on a series ofbiopsies of five different patients. Monoclonal envelope stainingpattern IGH 200 E1 negative IGH 201 E1 weak IGH 202 E1 negative IGH 204E1 weak IGH 207 E1 strong positivity of lymphocytes, weaker positivityof hepatocytes IGH 209 E1 negative IGH 210 E1 positive on lymphocytes(only noted after formaldehyde fixation) IGH 212 E2 weak IGH 214 E2negative IGH 215 E2 negative IGH 216 E2 negative IGH 219 E2 negative IGH221 E2 negative IGH 222 E2 strong positivity of hepatocytes

Example 3 Identification of Monoclonal Antibodies Allowing Detection ofViral Envelope Antigen in Peripheral Blood Cells

[0058] The finding that lymphocyte infiltrates in the liver can bestained for HCV envelope antigens prompted us to look also at peripheralblood mononuclear cells. In order to allow intracellular staining,peripheral blood cells were permeabilised with saponin, allowedthereafter to react with the monoclonal antibodies IGH 201 or 207(directed against E1 and showing weak and strong positivity on the liverbiopsies, respectively). Finally, reactivity was checked on afluorescent cell sorter using secondary FITC-labelled antibodies. IGH207, which stained already the lymphocyte infiltrates in the liver,showed a high specificity. With this monoclonal, almost no backgroundstaining was detected, and 4 out of 5 patients clearly stained positive(FIG. 3). The second monoclonal, IGH 201 (binding to SEQ ID 29; ECACCaccession number: 98031216) which is also directed against E1, yields ahigher background but intracellular E1 was detected in 5 out of 5patients as can be deduced from the histograms presented in FIG. 4,which show a clear subpopulation of cells with a higher degree offluorescence as compared to the control sample.

Example 4 Mapping of the Reactive Monoclonal Antibodies Against E1 or E2

[0059] All 14 monoclonal antibodies were mapped to their respectiveepitopes using peptides scanning the E1 and E2 protein against which theantibodies were raised. These peptides were biotinylated, bound tostreptavidin sensitized microtiterplates, and allowed to react with themonoclonal antibodies. As a positive control recombinant envelopeproteins were checked.

[0060] For each monoclonal antibody reactivity could be assigned to aspecific epitope region defined by two overlapping peptides (for detailson sequences see Table I). peptides aa region reactive monoclonals V1V2192-226 IGH 201, 204, 202, 200 V2V3 212-244 IGH 201, 204, 202, 200 V3V4230-263 HR 261-290 V5C4 288-327 IGH 207, 210, 209 C4V6 307-340 IGH 207,210, 209 recombinant 192-326 IGH 201, 207, 210, 204, 202, 200, 209 HVR I384-415 IGH 222,215 HVR1/C1a 395-428 IGH 222, 215 C1a 413-447 C1b430-467 HVRII 460-487 IGH 212, 214, 221 C2a 480-513 IGH 219, 216 C2b500-530 V3-C3 523-566 V4 561-590 C4a 578-627 C4b 621-648 C4c 641-673recombinant 384-673 IGH 222, 215, 212, 214, 221, 219, 216

[0061] The epitope for IGH 201 can be defined as the region 212-226 (SEQID 29), for IGH 207 and IGH 210 this is 307-326 (SEQ ID 30) and for IGH222 this is 395-415 (SEQ ID 31). The amino acid region of IGH 201 is arather variable region of the E1 protein of HCV and has already beenpreviously reported in relation to in situ detection of HCV (Hiramatsuet al., 1992, Kaito et al., 1994). However, from our studies it is clearthat antibodies directed against this epitope are less suitable for insitu detection of HCV as the liver biopsy staining with this antibodywas negative and the staining on peripheral blood lymphocytes showedconsiderable background. In contrast, IGH 207 and IGH 210 recognise acompletely conserved region of E1 (Maertens and Stuyver, 1997). Themonoclonal IGH 222 recognises a region of E2, which is part of theN-terminal hypervariable domain of E2, and proved to be very suitablefor efficient in situ detection of HCV.

Example 5 Determination of Cross-Reactivity on Variable Epitopes

[0062] Using an extended series of peptides derived from varioussequences of the N-terminal hypervariable epitope of E2, IGH 222 wasfurther characterised. Table 2 shows a summary of these experiments.From this table it can be concluded that this monoclonal reacts withseveral sequences, but fails to react with some others. Knowing thisepitope is sufficient for the man skilled in the art to raise additionalantibodies against this epitope with a better reactivity towards othersequences which are not recognised by IGH 222. Such sequences are by wayof example the peptides with #490, 940, 884, 484 and 494 but othersequences in the region between aa 395-415 may be found against whichIGH 222 may fail to react.

[0063] From these examples it is clear that the epitopes recognised bythe monoclonal antibodies IGH 201, 207, 210 and 222 are readilyaccessible for binding antibodies and allow detection of the antigen inliver biopsies and peripheral blood cells. The properties of themonoclonal antibodies IGH 201, 207, 210 and 222 are rather unique, sinceother monoclonals directed against the same epitopes resulted in highbackground and absence of specific staining. For example, the monoclonalantibodies IGH 207, 209 and 210 all recognise the same epitope, however,IGH 209 is not able to stain E1 antigen in any cell type, while IGH 210only stains lymphocytes and IGH 207 stains both lymphocytes andhepatocytes. With the instant invention, it should be feasible for theman skilled in the art to produce large series of antibodies (eitherpolyclonal or monoclonal of nature, in various species), that can beused for detection of natural HCV protein antigens.

[0064] The need to produce a series of antibodies against a givenepitope reflects the fact that the behaviour of an antibody is notexclusively determined by the epitope recognised but also by secondaryproperties such as affinity and avidity for the epitope, solubility,isotype, and the species in which it is generated. For each applicationan antibody with different secondary properties may be required. Thus,the production of such large series of antibodies will allow to identifysome of them as having similar properties as IGH 201, 207, 210 and 222i.e. to be able to reveal the presence of HCV envelope protein inbiological samples of the host. Indeed, the knowledge that theseepitopes are clearly accessible on the HCV envelope proteins, asexpressed in the host, provides the necessary information to developassays to detect these antigens not only in situ as presented here butalso in solution (eg in plasma or serum). The monoclonal antibodies IGH201, 207, 210 or 222, or others developed against the same epitope butwith optimal characteristics for detection of soluble antigen, possiblyin combination with others, could also be used to develop such an assay.

LIST OF REFERENCES

[0065] Cannon, E., Ladner R., and McCoy D. Phage-display technology. IVDTechnology November/December 1996,

[0066] Current protocols in immunology. Eds Coligan J., Kruisbeek A.,Margulis D., Shevach E., and Strober W. Wiley Interscience, 1992.

[0067] Dries V., Von Both I., Müller M., Gerken G., Schirmacher P.,Odenthal M., Bartenschlager R., Drebber U., Meyer Zum Büchenfelde K. -H.And Dienes H. P. Detection of Hepatitis C virus in paraffin-embeddedliver biopsies of patients negative for viral RNA in serum. Hepatology1999: 29: 223-229.

[0068] Guido M and Thung S. The value of identifying hepatitis C virusin liver pathology specimens. Hepatology 1996: 23: 376-379.

[0069] Hiramatsu N., Hayashi N., Haruna Y., Kasahara A., Fusamoto H.,Mori C., Fuke I., Okayama H. and Kamada T. Immunohistochemical detectionof hepatitis C virus-infected hepatocytes in chronic liver disease withmonoclonal antibodies to core, envelope and NS3 regions of the hepatitisC virus genome. Hepatology 1992: 16: 306-311.

[0070] Kaito M., Watanabe S., Tsukiyarna-Kohara K., Yamaguchi K.,Kobayashi Y., Konishi M., Yokoi M., Ishida S., Suzuki S. and Kohara M.Hepatitis C virus particle detected by immunoelectron microscopic study.J. Gen. Virol. 1994: 75: 1755-1760.

[0071] Kashiwakuma T., Hasegawa A., Kajita T., Takata A., Mori H., OhtaY., Tanaka E., Kiyosawa K., Tanaka T., Tanaka S., Hattori N. And KoharaM. Detection of hepatitis C virus specific core protein in serum ofpatients by a sensitive fluorescence enzyme immunoassay. J. Immunol.Meth. 1996: 190: 79-89.

[0072] Kohler and Milstein, (1975) Continuous cultures of fused cellssecreting antibody of predefined specificity. Nature 256: 495.

[0073] Liang, T. J. (1996) Hepatology elsewhere 23: 376-379.

[0074] Ladner, R. Constrained peptides as binding entities. Tibtech1995, 13:426-430.

[0075] Maclennan J. Engineering microprotein ligands for large-scaleaffinity purification. Biotechnology 1995, 13:1181-1183.

[0076] Maertens G. and Stuyver L. Genotypes and genetic variation ofhepatitis C virus. In: The molecular medicine of viral hepatitis. Ed:Harrison T. J. and Zuckerman A. J. 1997

[0077] Major M. E. and Feinstone S. M. The molecular virology ofhepatitis C. Hepatology 1997: 25: 1527-1538.

[0078] Sansonno D., Cornacchiulo V., Racanelli V. and Dammacco F. Insitu simultaneous detection of hepatitis C virus RNA and hepatitis Cvirus-related antigens in hepatocellular carcinoma. Cancer 1997: 80:22-33.

[0079] Sansonno D., Gesualdo L., Manno C., Schena F. and Dammacco F.Hepatitis C virus-related proteins in kidney tissue from hepatitis Cvirus-infected patients with cryoglobulinemic membranoproliferativeglomerulonephritis. Hepatology 1997: 25: 1237-1244. TABLE 1 E1 peptidesGenotype name # aa YQVRNSTGLYHVTNDCPNSSIVYEAADAILHTPGC 1a V1V2 1071192-226 YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGC 1b V1V2 888 192-226VEVKNNSNSYMATNDCSNSSIIWQLEGAVLHTPGC 2a V1V2 1019 192-226VEVKNTSTSYMVTNDCSNSSIVWQLEGAVLHTPGC 2c V1V2 1074 192-226LEWRNTSGLYVLTNDCSNSSIVYEADDVILHTPGC 3 V1V2 1008 192-226INYRNVSGIYHVTNDCPNSSIVYEADHHILHLPGC 4 V1V2 1075 192-226VPYRNASGIYHITNDCPNSSIVYEADNLILHAPGC 5 V1V2 1034 192-226LTYGNSSGLYHLTNDCSNSSIVLEADAMILHLPGC 6 V1V2 1023/1085 192-226LNYANKSGLYHLTNDCPNSSIVYEANGMILHLPG 7 V1V2 1334 192-225IQVKNASGIYHLTNDCSNSSIVFEAETMILHLPGC 9 V1V2 1333 192-226LEYRNASGLYMVTNDCSNGSIVYEAGDIILHLPGC 10 V1V2 1332 192-226IVYEAADMIMHTPGCVPCVRENNSSRCWV 1b V2V3 1036 212-244VRENNSSRCWVALTPTLAARNASVPTTTIRRHVD 1b V3V4 1022 230-263HVDLLVGAAAFCSAMYVGDLCGSVFLVSQL 1b HR 1150 261-290SQLFTISPRRHETVQDCNCSIYPGHITGHRMAWDMMNWS 1b V5C4 1176 288-327SIYPGHITGHRMAWDMMMNWSPTTALVVSQLLRI 1b C4V6 1039 307-340 E2 peptidesGenotype name # aa HTRVSGGAAASNTRGLVSLFSPGSAQKIQLVN 1b HVR I 1139384-415 NTRGLVSLFSPGSAQKIQLVNTNGSWHINRTALN 1b HVR I/C1a 1173 395-428LVNTNGSWHINRTALNCNDSLQTGFFAALFYKHKF 1b C1a 1149 413-447NDSLQTGFFAALFYKHKFNSSGCPERLASCRSIDKFAQ 1b C1b 1148 430-467RSIDKFAQGWGPLTYTEPNSSDQRPYCW 1b HVR II 1020 460-487SDQRPYCWHYAPRPCGIVPASQVCGPVYCFTPSP 1b C2a 1147 480-513SQVCGPVYCFTPSPVVVGTTDRFGVPTYNWG 1b C2b 1143 500-530GVPTYNWGANDSDVLILNNTRPPRGNWFGCTWWMGTGFTKTCGG 1b V3-C3 1178 523-566TKTCGGPPCNIGGAGNNTLTCPTDCFRKHP 1b V4 1142 561-590TDCFRKHPEATYARCGSGPWLTPRCMVHYPYRLWHYPCTVNFTIF 1b C4a 16 583-627TVNFTIFKVRMYVGGVEHRFEAACNWTR 1b C4b 1141 621-648EAACNWTRGERCDLEDPDRSELSPLLLSTTEWQ 1b C4c 1140 641-673KTTNRLVSMFASGPKQNVHLINT − HVR I 485 394-416 HTTSTLASLFSPGASQRIQLVNT −HVR I 492 395-416 HVTCTLTSLFRPGASQKIQLVNT − HVR I 489 394-416AHNARTLTGMFSLGARQKIQLINT − HVR I 520 394-416 SDTRGLVSLFSPGSAQKIQLVNT −HVR I 886 394-416 SSTQSLVSWLSQGPSQKIQLVNT − HVR I 494 394-416HTMTGIVRFFAPGPKQNVHLINT − HVR I 484 394-416 RAMSGLVSLFTPGAKQNIQLINT −HVR I 884 394-416 HVTGTLTSLFRPGASQKIQLVNT − HVR I 940 394-416

[0080]

1 41 1 35 PRT Hepatitis C virus 1 Tyr Glu Val Arg Asn Val Ser Gly IleTyr His Val Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Tyr GluAla Ala Asp Met Ile Met His Thr 20 25 30 Pro Gly Cys 35 2 29 PRTHepatitis C virus 2 Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr ProGly Cys Val 1 5 10 15 Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys TrpVal 20 25 3 34 PRT Hepatitis C virus 3 Val Arg Glu Asn Asn Ser Ser ArgCys Trp Val Ala Leu Thr Pro Thr 1 5 10 15 Leu Ala Ala Arg Asn Ala SerVal Pro Thr Thr Thr Ile Arg Arg His 20 25 30 Val Asp 4 30 PRT HepatitisC virus 4 His Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala MetTyr 1 5 10 15 Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu 2025 30 5 40 PRT Hepatitis C virus 5 Ser Gln Leu Phe Thr Ile Ser Pro ArgArg His Glu Thr Val Gln Asp 1 5 10 15 Cys Asn Cys Ser Ile Tyr Pro GlyHis Ile Thr Gly His Arg Met Ala 20 25 30 Trp Asp Met Met Met Asn Trp Ser35 40 6 34 PRT Hepatitis C virus 6 Ser Ile Tyr Pro Gly His Ile Thr GlyHis Arg Met Ala Trp Asp Met 1 5 10 15 Met Met Asn Trp Ser Pro Thr ThrAla Leu Val Val Ser Gln Leu Leu 20 25 30 Arg Ile 7 32 PRT Hepatitis Cvirus 7 His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asn Thr Arg Gly Leu1 5 10 15 Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu ValAsn 20 25 30 8 34 PRT Hepatitis C virus 8 Asn Thr Arg Gly Leu Val SerLeu Phe Ser Pro Gly Ser Ala Gln Lys 1 5 10 15 Ile Gln Leu Val Asn ThrAsn Gly Ser Trp His Ile Asn Arg Thr Ala 20 25 30 Leu Asn 9 35 PRTHepatitis C virus 9 10 38 PRT Hepatitis C virus 10 Asn Asp Ser Leu GlnThr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His 1 5 10 15 Lys Phe Asn SerSer Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser 20 25 30 Ile Asp Lys PheAla Gln 35 11 28 PRT Hepatitis C virus 11 Arg Ser Ile Asp Lys Phe AlaGln Gly Trp Gly Pro Leu Thr Tyr Thr 1 5 10 15 Glu Pro Asn Ser Ser AspGln Arg Pro Tyr Cys Trp 20 25 12 34 PRT Hepatitis C virus 12 Ser Asp GlnArg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly 1 5 10 15 Ile ValPro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro 20 25 30 Ser Pro13 31 PRT Hepatitis C virus 13 Ser Gln Val Cys Gly Pro Val Tyr Cys PheThr Pro Ser Pro Val Val 1 5 10 15 Val Gly Thr Thr Asp Arg Phe Gly ValPro Thr Tyr Asn Trp Gly 20 25 30 14 44 PRT Hepatitis C virus 14 Gly ValPro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile 1 5 10 15 LeuAsn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp 20 25 30 MetAsn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly 35 40 15 30 PRT Hepatitis Cvirus 15 Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn1 5 10 15 Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 20 2530 16 45 PRT Hepatitis C virus 16 Thr Asp Cys Phe Arg Lys His Pro GluAla Thr Tyr Ala Arg Cys Gly 1 5 10 15 Ser Gly Pro Trp Leu Thr Pro ArgCys Met Val His Tyr Pro Tyr Arg 20 25 30 Leu Trp His Tyr Pro Cys Thr ValAsn Phe Thr Ile Phe 35 40 45 17 28 PRT Hepatitis C virus 17 Thr Val AsnPhe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val 1 5 10 15 Glu HisArg Phe Glu Ala Ala Cys Asn Trp Thr Arg 20 25 18 33 PRT Hepatitis Cvirus 18 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp1 5 10 15 Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr GluTrp 20 25 30 Gln 19 23 PRT Hepatitis C virus 19 Lys Thr Thr Asn Arg LeuVal Ser Met Phe Ala Ser Gly Pro Lys Gln 1 5 10 15 Asn Val His Leu IleAsn Thr 20 20 23 PRT Hepatitis C virus 20 His Thr Thr Ser Thr Leu AlaSer Leu Phe Ser Pro Gly Ala Ser Gln 1 5 10 15 Arg Ile Gln Leu Val AsnThr 20 21 23 PRT Hepatitis C virus 21 His Val Thr Cys Thr Leu Thr SerLeu Phe Arg Pro Gly Ala Ser Gln 1 5 10 15 Lys Ile Gln Leu Val Asn Thr 2022 24 PRT Hepatitis C virus 22 Ala His Asn Ala Arg Thr Leu Thr Gly MetPhe Ser Leu Gly Ala Arg 1 5 10 15 Gln Lys Ile Gln Leu Ile Asn Thr 20 2323 PRT Hepatitis C virus 23 Ser Asp Thr Arg Gly Leu Val Ser Leu Phe SerPro Gly Ser Ala Gln 1 5 10 15 Lys Ile Gln Leu Val Asn Thr 20 24 23 PRTHepatitis C virus 24 Ser Ser Thr Gln Ser Leu Val Ser Trp Leu Ser Gln GlyPro Ser Gln 1 5 10 15 Lys Ile Gln Leu Val Asn Thr 20 25 23 PRT HepatitisC virus 25 His Thr Met Thr Gly Ile Val Arg Phe Phe Ala Pro Gly Pro LysGln 1 5 10 15 Asn Val His Leu Ile Asn Thr 20 26 23 PRT Hepatitis C virus26 Arg Ala Met Ser Gly Leu Val Ser Leu Phe Thr Pro Gly Ala Lys Gln 1 510 15 Asn Ile Gln Leu Ile Asn Thr 20 27 23 PRT Hepatitis C virus 27 HisVal Thr Gly Thr Leu Thr Ser Leu Phe Arg Pro Gly Ala Ser Gln 1 5 10 15Lys Ile Gln Leu Val Asn Thr 20 28 23 PRT Hepatitis C virus 28 Arg ThrThr Gln Gly Leu Val Ser Leu Phe Ser Arg Gly Ala Lys Gln 1 5 10 15 AspIle Gln Leu Ile Asn Thr 20 29 15 PRT Hepatitis C virus 29 Ile Val TyrGlu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys 1 5 10 15 30 20 PRTHepatitis C virus 30 Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met AlaTrp Asp Met 1 5 10 15 Met Met Asn Trp 20 31 21 PRT Hepatitis C virus 31Asn Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys 1 5 1015 Ile Gln Leu Val Asn 20 32 35 PRT Hepatitis C virus 32 Tyr Gln Val ArgAsn Ser Thr Gly Leu Tyr His Val Thr Asn Asp Cys 1 5 10 15 Pro Asn SerSer Ile Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr 20 25 30 Pro Gly Cys35 33 35 PRT Hepatitis C virus 33 Val Glu Val Lys Asn Asn Ser Asn SerTyr Met Ala Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Ile Trp GlnLeu Glu Gly Ala Val Leu His Thr 20 25 30 Pro Gly Cys 35 34 35 PRTHepatitis C virus 34 Val Glu Val Lys Asn Thr Ser Thr Ser Tyr Met Val ThrAsn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Trp Gln Leu Glu Gly AlaVal Leu His Thr 20 25 30 Pro Gly Cys 35 35 35 PRT Hepatitis C virus 35Leu Glu Trp Arg Asn Thr Ser Gly Leu Tyr Val Leu Thr Asn Asp Cys 1 5 1015 Ser Asn Ser Ser Ile Val Tyr Glu Ala Asp Asp Val Ile Leu His Thr 20 2530 Pro Gly Cys 35 36 35 PRT Hepatitis C virus 36 Ile Asn Tyr Arg Asn ValSer Gly Ile Tyr His Val Thr Asn Asp Cys 1 5 10 15 Pro Asn Ser Ser IleVal Tyr Glu Ala Asp His His Ile Leu His Leu 20 25 30 Pro Gly Cys 35 3735 PRT Hepatitis C virus 37 Val Pro Tyr Arg Asn Ala Ser Gly Ile Tyr HisIle Thr Asn Asp Cys 1 5 10 15 Pro Asn Ser Ser Ile Val Tyr Glu Ala AspAsn Leu Ile Leu His Ala 20 25 30 Pro Gly Cys 35 38 35 PRT Hepatitis Cvirus 38 Leu Thr Tyr Gly Asn Ser Ser Gly Leu Tyr His Leu Thr Asn Asp Cys1 5 10 15 Ser Asn Ser Ser Ile Val Leu Glu Ala Asp Ala Met Ile Leu HisLeu 20 25 30 Pro Gly Cys 35 39 34 PRT Hepatitis C virus 39 Leu Asn TyrAla Asn Lys Ser Gly Leu Tyr His Leu Thr Asn Asp Cys 1 5 10 15 Pro AsnSer Ser Ile Val Tyr Glu Ala Asn Gly Met Ile Leu His Leu 20 25 30 Pro Gly40 35 PRT Hepatitis C virus 40 Ile Gln Val Lys Asn Ala Ser Gly Ile TyrHis Leu Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Phe Glu AlaGlu Thr Met Ile Leu His Leu 20 25 30 Pro Gly Cys 35 41 35 PRT HepatitisC virus 41 Leu Glu Tyr Arg Asn Ala Ser Gly Leu Tyr Met Val Thr Asn AspCys 1 5 10 15 Ser Asn Gly Ser Ile Val Tyr Glu Ala Gly Asp Ile Ile LeuHis Leu 20 25 30 Pro Gly Cys 35

1. Use of antibodies specifically binding to the C-terminal region ofthe HCV E1 protein (aa 227-383) or the N-terminal region of the HCV E2protein (aa 384-450) for the preparation of a natural HCV proteinantigen detection kit.
 2. The use of antibody according to claim 1wherein said antibody specifically binds to one of the followingepitopes: aa 307-326 of HCV E1 protein (SEQ ID 30) aa 395-415 of HCV E2protein (SEQ ID 31).
 3. The use of antibodies according to any of claims1 or 2 wherein said antibody is a monoclonal antibody.
 4. The use ofantibodies according to any of claims 1 to 3 wherein said antibody issecreted by ECACC deposit having accession number 98031214 or 98031215.5. The use of a functionally equivalent variant or fragment of anantibody according to any of claims 1 to
 4. 6. The monoclonal antibodysecreted by the ECACC deposit having accession number
 98031214. 7. Thefunctionally equivalent variant or fragment of the antibody according toclaim
 6. 8. The hybridoma cell line of ECACC deposit having accessionnumber 98031214
 9. Method for the detection of natural HCV proteinantigens comprising: contacting a test sample which may contain HCVprotein antigens with an antibody according to any of claims 1 to 4 orwith a functionally equivalent variant or fragment of said antibody, toform an antibody-antigen complex, and determining said antigen-antibodycomplex with an appropriate marker.
 10. The method according to claim 9,wherein said test sample comprises human cells or tissues.
 11. Themethod according to claim 10, wherein said human cells are peripheralblood cells.
 12. The method according to claim 10, wherein said humantissue is liver tissue.
 13. An assay kit for the detection of naturalHCV protein antigens comprising: an antibody according to any of claims1 to 4, or a functionally equivalent variant or fragment of saidantibody, and appropriate markers which allow to determine the complexesformed between HCV protein antigens in a test sample with said antibodyor a functionally equivalent variant or fragment of said antibody.